We directly observed charge separation and a space‐charge region in an organic single‐crystal p–n heterojunction nanowire, by means of scanning photocurrent microscopy. The axial p–n heterojunction nanowire had a well‐defined planar junction, consisted of P3HT (p‐type) and C60 (n‐type) single crystals and was fabricated by means of the recently developed inkjet‐assisted nanotransfer printing technique. The depletion region formed at the p–n junction was directly observed by exploring the spatial distribution of photogenerated carriers along the heterojunction nanowire under various applied bias voltages. Our study provides a facile approach toward the precise characterization of charge transport in organic heterojunction systems as well as the design of efficient nanoscale organic optoelectronic devices.